556 SHORT COMMUNICATIONS

~BA 23369

Selective nucleolar uptake of ~l-acid glycoprotein by mammalian cells in tissue culture

In earlier papers from this laboratory 1,2 we reported a new chemically defined medium, CMRL-I415, and described the preparation and use of certain supplements for this medium. When the supernatant solution of Cohn's fraction V from human plasma (method 6), sometimes referred to as fraction VI, was used as a supplement for CMRL-I415, there was marked improvement in mouse embryo cultures, especially when it was used together with dextran (mol. wt. 1ooooo-2o0oo0). Supernatant V was further fractionated by gel filtration to give a 3-fold concentration of growth activity in a single highly purified fraction that proved to be ~l-acid glycoprotein, or orosomucoid 2. Experiments with component subunits of ~l-acid glycoprotein that were not present in the basal medium (fucose, sialic acid, mannose and A:-acetyl- glucosamine) revealed that these components alone or in combination with dextran were without effect on the growth of the cells. Fluorescein isothiocyanate was conju- gated with ~z-acid glycoprotein, and the conjugate was incorporated in CMRL-I4I 5 for the cultivation of several mammalian cell types to discover if the labeled protein had the same effect as native ~z-acid glycoprotein and whether labeled material entered the cells during cultivation. I t is the purpose of the present report to show that fluorescein isothiocyanate coupled with cq-acid glycoprotein behaves like native ~t-acid glycoprotein and is taken up selectively by the cell nucleoli.

Supernatant V was concentrated, dialysed and dried in vacuo from the frozen state, as described earlier 2. cq~Acid glycoprotein was separated from a solution of this mixture by chromatography on carboxymethyl cellulose s. The isolated protein was shown to be homogeneous by zone electrophoresis in acrylamide gels and by agar-gel immunoelectrophoresis. The isolated protein was then coupled with fluo- rescein isothiocyanate by the procedure of RINDERKNECHT 4. After removal of the excess unreacted dye, the fluorescent-labeled ~-acid glycoprotein was lyophilized.

The cells used in this investigation included Earle's L cells (mouse), Gey's HeLa cells (human), a line of dog kidney cells isolated in this laboratory, and newly isolated mouse embryo cells. The cultures were prepared by methods comparable to those described earlier ~, but for these experiments I ml of test medium was inoculated into Leighton tubes containing coverslips. Replicate cultures containing unlabeled protein (control) and others containing labeled protein (test) were incubated at 37 ° in s tat ionary tubes for periods up to IO days, with complete fluid changes twice a week. At intervals, one control and two test cultures were terminated and prepared for ultraviolet fluorescence microscopy. In some experiments, test culture fluids were replaced by control (unlabeled) fluids after 3 days in order to estimate the degree of turnover of any labeled protein that might have been incorporated. When the cultures were terminated, the medium was decanted, the tubes were filled with warm phos- phate buffered saline, then filled for I min with IO % aqueous glutaraldehyde and finally washed twice with distilled water. The coverslips were then removed, mounted on standard microscope slides with I : I veronal buffer prepared with neutral glycerine, and the preparations were sealed by painting the margins of the coverslips with clear lacquer. Fluorescence microscopy was carried out with a Reichert Zetopan microscope fitted with contrast fluorescence equipment and an automatic camera.

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SHORT COMMUNICATIONS 557

Although it was anticipated that cells might incorporate discrete droplets of labeled material into the cytoplasm, as observed by EASTY, YAR.~'ELL A~D ANDREWS 5 with certain proteins labeled with fluorescein, preliminary experiments revealed an entirely different phenomenon. The presence of labeled c~l-acid glycoprotein in the culture fluids resulted in an intense apple-green fluorescence in the nucleoli of all cells tested. The bright fluorescence was apparent after 24 h (Fig. I), reached a maximum in 48 h, and remained in the nucleoli of healthy cells as long as labeled protein was present in the medium. The cytoplasm of all cells revealed only blue autofluorescence during the entire period of cultivation. If, after 3 days' cultivation, the control (unlabeled) medium was used to replace the test medium, the green fluorescence decreased and disappeared upon further cultivation.

Fig. I. Fluorescent image of an altered dog kidney cell (from 9-year-old line) cultivated in CMRL- 1415 supplemented with fluorescent cq-acid glycoprotein. Note the four bright nucleoli, Magni- fication: × 27oo.

The removal of protein from the medium by cells occurs in two phases, an initial rapid adsorption to the cell surface and a slower incorporation by pinocytosis6,L The vacuoles proceed inwards and become smaller and more concentrated as they approach the nucleus 8. Evidence exists from ultrastructure studies that the pores of the nuclear membrane are important pathways for macromolecular exchanges between the cell cytoplasm and the nucleoplasm °. CooNs, LEDUC AND KAPLAN 10 and MAYERSBACH AND PEARSE 11 traced injected foreign proteins in vivo in mice. By fluo- rescent antibody techniques they often found antigen in higher concentration in nuclei

Biochim. Biophys. Acta, 148 (1967) 556-558

558 SHORT COMMUNICATIONS

than in the cytoplasm; but the fluorescence was confined to the nuclear sap and was not present in the nucleoli. Nucleolar fluorescence has been reported by ~V[AISEL 12 and by MAISEL a ~ ' o LYTLE 13, who found by means of a nonimmune fluorescence technique that nucleolar fluorescence is produced in monolayers of cells by a con> ponent of animal sera. The active material was shown to be an ~-globulin (glyco- protein), but %-acid glycoprotein from Cohn's fraction VI was not effective in producing nucleolar fluorescence in their system.

Earlier, in this laboratory, the disappearance of %-acid glycoprotein from the medium was assumed to be an index of uptake of this protein by mouse embryo cells". The appearance of the same fluorescent labeled protein in the nucleoli of cells culti- vated under the same conditions is direct evidence that this protein enters the cells. The method does not rule out the possibility that only a portion of the glycoprotein molecule may have been incorporated.

This study was supported, in part, by a grant from the Donner Canadian Foundation.

Connaught Medical Research Laboratories, University o[ Toronto, Toronto (Canada)

GEORGE M. HEALY

RAYMOND C. PARKER

I G. 3~. HEALY AND R. C. PARKER, J. Cell Biol., 30 (1966) 531. 2 G. M. HEALY AND ]~. C. PARKER, J. Cell Biol., 30 (1966) 539. 3 A. BEZKOROVAINY AND R. J, WINZLER, Biochim. Biophys. Acta, 49 (I96I) 559. 4 1-t. RINDERKNECHT, Nature, 193 (1962) 167. 5 G. C. EASTY, M. M. YARNELL AND R. D. ANDREWS, Brit. J. Cancer, 18 (1964) 354. 6 W. H. LEWIS, Am. J. Cancer, 29 (I937) 666. 7 H. J . -P . RYSER, Lab. Invest., 12 (1963) lOO9. 8 G. O. GEY, Harvey Lectures Ser., 5 ° (1954-1955) 154. 9 C. M. FELDHERR, J. Cell Biol., 25 (1965) 43,

io A. H. CooNs, g . H. LEDUC AND M. H. KAPLAN, J. Exptl. Med., 93 (1951) 173. i i }{. MAYERSBACH AND A. G. E. PEARSE, Bril. J. Exptl. Pathol., 37 (1956) 81. i2 J. C. MAISF,,L, J. Lab. Clin. Med., 60 (I962) 357. 13 J. C. MAISEL AND R. I. LYTLE, J. Cell Biol., 29 (I966) 461.

Received July Ioth, 1967

Biochim. Biophys. Acta, 148 (1967) 556--558

BBA 23367 Evidence for excretion of certain enzymes during growth of protoplasts of Bacillus megaterium

It is well known that exoenzymes, such as ribonuclease (EC 2.7.7.16) and c~-amylase (EC 3.2.1.z), are released from bacterial cells cultured in either liquid 1,2 or solid media a. However, nothing is yet known about how the enzymes are released from the cells. Practically no work has been done on the problem of enzyme liberation because of certain characteristics of bacterial cells. Bacterial cells are easily lysed in resting or sometimes even in growing conditions and the lysed cells have been

Abbrev ia t ion : DCIP, 2 ,6-dichlorophenol indophenol .

Biochim. Biophys. Acta, 148 (1967) 558-560